We study apoptosis (programmed cell death) in cultured rat (and soon mouse) neurons. We will study the physiology of mitochondria during apoptosis using nonyl acridine orange (NAO), which specifically stains cardiolipin, a lipid found only in mitochondria. In order to view those dying cells which no longer stain with NAO, we will counterstain with Hoechst. By double-labeling, we can quantify the NAO staining and demonstrate the loss of mitochondrial labeling in cells that are near death. We also plan to use other dyes to study other aspects of mitochondrial integrity, such as membrane potential using Rhodamine 123. We will still need to counterstain the nuclei in these studies if mitochondria fail to stain in the late stages of apoptosis.